Multidrug-resistant tuberculosis (MDR-TB), defined as Mycobacterium tuberculosis (Mtb) resistant to the two first-line drugs isoniazid and rifampin, has emerged as a major threat to global TB control. The situation is particularly acute in China, which is one of 27 high-burden MDR-TB countries. In >95% of clinical MDR-TB isolates, rifampin resistance is mediated by mutations in a relatively short segment of the rpoB gene, which encodes the molecular target of rifampin. Although laboratory-derived mutants with a variety of different rpoB gene mutations show slower growth under nutrient-rich conditions, Mtb clinical strains isolated from individuals with TB who developed rifampin resistance during treatment show normal growth compared to their rifampin-susceptible counterparts, despite harboring the same mutation as some of the laboratory-derived strains. Recently, we have found evidence that a known lipid virulence factor, phthiocerol dimycocerosate (PDIM), accumulates in the cell wall of rifampin-resistant TB organisms. Studies using laboratory-generated double mutant strains revealed that PDIM is required for the normal survival of rifampin-resistant strains in activated mouse macrophages. The central hypothesis of this proposal is that the induction of pathways involved in biosynthesis and transport of cell wall-associated lipid virulence factors is a compensatory metabolic adaptation, which serves to enhance the virulence of rifampin-resistant clinical isolates in the infected host. This proposal represents a unique collaboration between investigators at Jiao Tong University School of Medicine in Shanghai, China and Johns Hopkins University School of Medicine in Baltimore, U.S.A. Using a multidisciplinary approach, including the use of a novel mouse model, which develops TB lung lesions resembling their human counterparts, in combination with transcriptional, lipidomic, genetic, and imaging techniques, we will investigate whether PDIM accumulation compensates for the fitness cost associated with Mtb rpoB mutation during host infection. Our data are expected to yield novel drug targets, with the ultimate goal of shortening the duration of MDR-TB treatment in China and worldwide.